**7.2. Regulation of the biosynthesis of the core**

78 The Complex World of Polysaccharides

**7. Regulation of the expression of LPS** 

and, in some organisms, in plasmids [22,23].

acid in the biosynthesis of phospholipids [10].

are coded in constitutive genes.

and 52.7%, respectively.

the regulon *phoP-phoQ* [25].

pathogenicity islands (PAI) [10,24].

**7.1. Regulation of the biosynthesis of lipid A** 

The biosynthesis of LPS is performed through two separate pathways. One pathway involves the formation of lipid A and the core, and the other pathway involves the formation of the O antigen. In the synthesis of LPS, a large number of genes participate, many of which are part of clusters located in different regions of the bacterial chromosome

Lipid A and the core oligosaccharide are formed in a continuous process, which is separate from the synthesis of the O antigen. In the majority of *Enterobacteriaceae*, the genes involved in this synthesis are found in a single copy and share several characteristics with

The genes involved in the first steps of the biosynthesis of lipid A in *E. coli, S. enterica*  serovar Typhimurium, *Yersiia enterocolitica*, *Haemophilus influenzae* and *Rickettsia rickettsii* are grouped into the *lpx*D-*fab*Z*-lpx*A-*lpx*B cluster[8]. The genes *lpx*A and *lpx*D code for Nacyltransferases, which add fatty acids to the glucosamine disaccharide. Both enzymes contain a conserved repeated structure, which is the hexapeptide [(I,V,L)GXXXX]n. The gene *lpx*B is a co-transcript with *lpx*A and codes for the disaccharide synthase of lipid A, which catalyses the formation of the disaccharide of lipid A from UDP-2.3-diacylglucosamine and 2,3-diacylglucosamine-1-phosphate. The gene *fab*Z codes the enzyme that catalyses the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, which is used as a donor of fatty

The proteins involved in the biosynthetic pathways of UDP-GlcNAc, UDP-Glc and UDP-Gal

L-glycero-D-manno-heptose is added to its derivative ADP, which is synthesised from sedoheptulose 7-phosphate in four steps. The genes *gmhA* and *gmhD* code for enzymes in the first and last steps. The G+C content of the *gmhA*, *gmhD* and *waaE* genes are 51%, 51%

Kdo is transferred from CMP-Kdo and synthesised from arabinose-5P and PEP by a threestage pathway[9]. Two of the genes in this process, namely, *kdsA* and *kdsB*, are well-

The gene *waaA*, which is located in the *waa* cluster, codes for a bifunctional Kdo transferase that adds residues from 2 Kdo. These genes have a G+C content of between 51% and 54%. Several of the modifications of lipid A are regulated by the concentration of Mg2+ through

PhoP-PhoQ is a two-component system that regulates virulence through adaptation to limited magnesium environments and regulates numerous cellular activities in gramnegative bacteria. This regulon consists of an external membrane sensor, PhoQ, and a cytoplasmic regulator, PhoP, and is activated by the acidic pH and by certain antimicrobial

characterised and have G+C contents of 51.6% and 52.7%, respectively.

In *E. coli*, *Salmonella* and *K. pneumoniae*, the genes involved in the biosynthesis of the core are grouped on the chromosome. These loci code for the activities required in the assembly of the outer core and also code for the transferases necessary for the synthesis of the inner core. In *E. coli* and *Salmonella*, the *waa* locus (also called *rfa*) is formed by three operons and is located between the genes *cysE* and *pyrE*. The operons are defined by the name of the first gene in each transcriptional unit, such as *gmhD, waaQ* and *waaA* (**Figure 3**) [28].

Genes in the *waa* operon code for all of the transferases that assemble the core, including the gene that codes for the enzyme for the last step of the synthesis of ADP-L-Glycero-Dmanno-heptose and the gene of the ligase of the O antigen, *waaL*. In addition, the *waa* operon contains the gene *waaA*, which codes for a bifunctional transferase Kdo.

The operon *gmhD*, which is located on the extreme 5' end of the *waa* cluster, contains *gmhD, waaF, waaC* and *waaL*. The genes *gmhD*-*waaFC* are required for the biosynthesis and transfer of L, D-heptose. GmhD catalyses the last reaction of the synthesis of ADP-L-Glycero-Dmanno-heptose. WaaC is the transferase for Hepl, and WaaF is a putative transferase of Hepll. WaaL is the ligase that bonds the O polysaccharide to the lipid A and core.

The transcription of the operon *gmhD* in *E. coli* K12 is regulated by heat shock promoters, indicating a requirement for the heptose domain of LPSs for growth at high temperatures [23,28,29].

The central operon *waaQ* contains 10 genes that are necessary for the biosynthesis of the outer core and for the modification of the core. WaaQ is the transferase for HepIII. The proteins WaaG, WaaO and WaaR are transferases for Glcl, Glcll and GlcIII, respectively, and WaaB is the transferase for the GaL residue. WaaP and WaaY are involved in the phosphorylation of the residue of heptose, whereas the functions of WaaU, WaaS and WaaZ are still not clear. In isolates of *E. coli* with the R1 and R4 cores, this operon contains the structural gene of ligase *waaL*, which must be produced for the union of the O polysaccharide with the complete core.

The *waaQ* operon is preceded by a JUMPStart sequence (Just Upstream of Many Polysaccharide-associated gene Starts), which includes a conserved region of 8 bp that is known as *ops* (operon polarity suppressor). The expression of the cluster for the biosynthesis of the core is regulated by the protein RfaH and also in response to thermal shock. RfaH is homologous to the factor NusG, which regulates the expression of the operon of hemolysin, genes of the polysaccharides of the capsule and genes for the transfer of the F plasmid. Regulation by RfaH occurs at the level of the polymerisation of the mRNA and depends on *ops* sequences that act in *cis*, as in the case of the operon *waaQGPSBIJYZK*, which includes 10 genes of the cluster of the core of LPS [22,30].

The transcript *waaA* contains the structural gene *waaA* (formally called *kdtA*), which codes for the transferase Kdo and a "non LPS" gene that codes for the adenylyltransferase pantetheine (*coaD*, formally *kdtB*) [31].

**Figure 3.** Structure of the genes involved in the synthesis of the core of *E. coli* R1. The genes of the glycosyltransferases that form the bonds of the inner core are shown in green. Those genes of the enzymes that modify the structure are in red, and the glycosyltransferases of the outer core are in blue. In orange are genes that modify the ligase enzyme.

## **7.3. Regulation of the biosynthesis of the O polysaccharide**

The genes involved in the biosynthesis of the O antigen are generally found in the chromosome in the cluster of the O antigen or *rfb*. These genes have a lower GC content than the average for genomes, between 30% and 40%, which provides evidence that these genes have been acquired through lateral inter-species transference [24].

The genes that code for the proteins that participate in the synthesis of the O antigen form three main groups: a) proteins involved in the biosynthesis of the precursors of nucleotide sugars of the O antigen; b) protein glycosyltransferases, which sequentially transfer various precursor sugars to form an oligosaccharide of a lipid carrier, undecaprenyl phosphate (UndP), which is located in the cytoplasmic face of the internal membrane and c) genes for the processing of the O antigen, which are involved in the translocation through the membrane and polymerisation (**Figure 4**) [32,33]. A fraction of O antigens includes acetyl-O groups, and others include residues; therefore, in the corresponding clusters, the transferases for them are coded. The differences among the many forms of the O antigen are due to the genetic variation in the cluster of the O antigen. The genes for the initial steps, which are also involved in conserved functions, do not duplicate in the cluster of the O antigen [24].

Mechanisms of O-Antigen Structural Variation of Bacterial Lipopolysaccharide (LPS) 81

80 The Complex World of Polysaccharides

genes of the cluster of the core of LPS [22,30].

In orange are genes that modify the ligase enzyme.

antigen [24].

**7.3. Regulation of the biosynthesis of the O polysaccharide** 

have been acquired through lateral inter-species transference [24].

pantetheine (*coaD*, formally *kdtB*) [31].

The *waaQ* operon is preceded by a JUMPStart sequence (Just Upstream of Many Polysaccharide-associated gene Starts), which includes a conserved region of 8 bp that is known as *ops* (operon polarity suppressor). The expression of the cluster for the biosynthesis of the core is regulated by the protein RfaH and also in response to thermal shock. RfaH is homologous to the factor NusG, which regulates the expression of the operon of hemolysin, genes of the polysaccharides of the capsule and genes for the transfer of the F plasmid. Regulation by RfaH occurs at the level of the polymerisation of the mRNA and depends on *ops* sequences that act in *cis*, as in the case of the operon *waaQGPSBIJYZK*, which includes 10

The transcript *waaA* contains the structural gene *waaA* (formally called *kdtA*), which codes for the transferase Kdo and a "non LPS" gene that codes for the adenylyltransferase

JUMP start site

**Figure 3.** Structure of the genes involved in the synthesis of the core of *E. coli* R1. The genes of the glycosyltransferases that form the bonds of the inner core are shown in green. Those genes of the enzymes that modify the structure are in red, and the glycosyltransferases of the outer core are in blue.

*gmhD waaF waaC waaL waaV waaW waaY waaT waaO waaP waaG waaO waaA*

The genes involved in the biosynthesis of the O antigen are generally found in the chromosome in the cluster of the O antigen or *rfb*. These genes have a lower GC content than the average for genomes, between 30% and 40%, which provides evidence that these genes

The genes that code for the proteins that participate in the synthesis of the O antigen form three main groups: a) proteins involved in the biosynthesis of the precursors of nucleotide sugars of the O antigen; b) protein glycosyltransferases, which sequentially transfer various precursor sugars to form an oligosaccharide of a lipid carrier, undecaprenyl phosphate (UndP), which is located in the cytoplasmic face of the internal membrane and c) genes for the processing of the O antigen, which are involved in the translocation through the membrane and polymerisation (**Figure 4**) [32,33]. A fraction of O antigens includes acetyl-O groups, and others include residues; therefore, in the corresponding clusters, the transferases for them are coded. The differences among the many forms of the O antigen are due to the genetic variation in the cluster of the O antigen. The genes for the initial steps, which are also involved in conserved functions, do not duplicate in the cluster of the O **Figure 4.** The synthesis of the O antigen involves three types of genes: a) genes related to the biosynthesis of precursors of sugar (green boxes), whose protein products perform their function in the cytoplasm; b) genes for glycosyltransferases (blue boxes), whose corresponding proteins transfer nucleotide sugars in the UndP lipid to form the O unit. This process occurs on the cytoplasmic side of the internal membrane and c) genes for the assembly of the O antigen and its exportation (orange box). Modified from [38].

In *E. coli* and *S. enterica*, the genes involved in the synthesis of the O antigen are typically found among the constituent genes *galF* and *gnd*. In *P. aeruginosa,* these genes are found among *himD* and *tyrB* [15]). In *V. cholerae*, O antigen synthesis genes are found among *gmhD*  and *rjg* [34], and in *Yersinia* spp., these genes are found among *hemH* and *gsk.* Some exceptions, such as the polysaccharide O54 of *S. enterica*, which is a cluster that is coded in a plasmid [35] (**Figure 5A**).

The genes of the biosynthetic pathway of the three precursors of the nucleotides of sugar are grouped within the genetic cluster of the O antigen. *manB and manC* code for enzymes that convert mannose-6-P into GDP-mannose. The operon *rmlABCD* codes for enzymes that form TDP-rhamnose from glucose-1-P. *ddhABCD* and *abe* code for enzymes to make CDP-abequose from glucose 1-P. UDP-Gal is used in other pathways and is synthesised by conserved enzymes. The transferase galactose, which is coded by *wbaP*, initiates the synthesis of the O units by transferring galactose phosphate from UDP-Gal to UndP.

The transferases coded by *wbaZ*, *wbaW* and *wbaQ* are positioned over two residues of mannose and one of rhamnose before the residues of mannose, which is acetylated by WbaL. The residue of abequose is bonded to acetyl rhamnose by WbaR to form a complete O unit. The flippase of the bond of the O antigen is coded by *wzx* and is responsible for

moving the O unit bonded to UndP through the internal membrane before the polymerase Wzy exerts its action over the long-chain O antigen.

The majority of the O antigen operons are constitutively expressed and are preceded by a sequence of 39 bp, known as JUMPStart **(Figure 5B)**.This sequence includes two elements known as *ops*, which are involved in the recruiting of elongation factors during the transcription. The JUMPStart region is controlled by RfaH, which acts as a positive regulator for the genes involved in the outer core of LPS and also increases the expression of the locus *rfb* [1,18,19*.*]

**Figure 5. A.** The location of the genes involved in the synthesis of the O antigen in several species of *Enterobacteriaceae*. **B.** The JUMPStart sequence of *S. flexneri* strain IB1833**.** The *ops* sequence is indicated in bold for eight base pairs. A residue of thymine that is highly conserved in the far 3' is indicated by cursive text, and repeated sequences are underlined. Accession number: U118168.1.
